Graduate Thesis Or Dissertation
 

Selenomethionine and methionine in in vitro rabbit reticulocyte hemoglobin synthesis and in Escherichia coli tRNA aminoacylation

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  • Selenomethionine and methionine have been postulated to be similarly involved in protein synthesis. This thesis has evaluated this hypothesis from a molecular and genetic approach. Selenomethionine and methionine were incorporated into rabbit reticulocyte hemoglobin at the same rates and in equal quantities. Each methionine demonstrated competitive inhibition for its analog incorporation into the globin. There were no significant differences in a or (3 chains for the methionine or selenomethionine rates of incorporation. Ion-exchange peptide analysis from a tryptic hydrolysate from this globin showed that selenomethionine and methionine were probably incorporated into the same peptides. However the peptides containing selenomethionine were somewhat displaced from the peptides containing methionine. When methyl labeled methionine and selenium labeled selenomethionine were incorporated into the globin, both their activities could be reduced 56-76% with dialysis against sodium sulfite. It was concluded that this sulfitolysis procedure was removing activity from the methionine and selenomethionine residues in the globin. There was no difference in selenomethionine or methionine aminoacylation of tRNA using materials from normal or selenium adapted Escherichia coli B. Both methionine and selenomethionine were rapidly bound in this system. Assay by plating on membrane filters demonstrated a higher binding of selenomethionine than methionine. However, assays by reverse phase chromatography showed that methionine and selenomethionine were bound in equal amounts and to the same two tRNA species. The two methionines were competitive inhibitors of each other for the aminoacylation of tRNA. Aminoacyl synthetase and tRNA were then prepared free of ribonuclease. Both selenomethionine and methionine had about the same optimum aminoacylation conditions: enzyme and tRNA concentration, ATP/Mg ratios, and pH range. Both aminoacylations were completely dependent upon added ATP. Sodium ion inhibited both aminoacylations, but could be overcome by potassium ion. Selenomethionine was aminoacylated faster and in preference to methionine, but showed no saturation of tRNA when selenomethionine concentration was increased. Aminoacylated selenomethionine- and methionine-tRNA could be deaminoacylated at pH 8.8, and then reaminoacylated to the same degree. Reverse phase column chromatography indicated that from this ribonuclease free material, there was only one methionine accepting tRNA species, which was identical in position and stoichiometry to the selenomethionine accepting tRNA species.
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