Abstract
The aminoacylation of rat liver tRNA with selenocysteine was studied in tissue slices and in a cell-free system with [75Se]selenocysteine and [75Se]selenite as substrates. [75Se]Selenocysteyl tRNA was isolated via phenol extraction, 1 M NaCl extraction and chromatography on DEAE-cellulose. [75Se]Selenocysteyl tRNA was purified on columns of DEAE-Sephacel, benzoylated DEAE-cellulose and Sepharose 4B. In a dual-label aminoacylation with [35S]cysteme, the most highly purified 75Se-fractions were > 100-fold purified relative to 35S. These fractions contained < 0.7% of the [35S]cysteine originally present in the total tRNA. When [35Se]selenocysteyl tRNA was purified from a mixture of 14C-labeled amino acids, over 97% of the [14C]aminoacyl tRNA was removed. The [75Se]selenocysteine was associated with the tRNA via an aminoacyl linkage. Criteria used for identification included alkaline hydrolysis and recovery of [75Se]selenocysteine, reaction with hydroxylamine and recovery of [75Se]selenocysteyl hydroxamic acid and release of 75Se by ribonuclease. The specificity of [75Se]selenocysteine aminoacylation was demonstrated by resistance to competition by a 125-fold molar excess of either unlabeled cysteine or a mixture of the other 19 amino acids in the cell-free selenocysteine aminoacylation system.
Original language | English (US) |
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Pages (from-to) | 183-191 |
Number of pages | 9 |
Journal | BBA - Gene Structure and Expression |
Volume | 699 |
Issue number | 3 |
DOIs | |
State | Published - Dec 31 1982 |
Keywords
- (Rat liver)
- Aminoacylation
- Selenocysteine
- tRNA
ASJC Scopus subject areas
- Biochemistry
- Biophysics
- Genetics
- Structural Biology
- Medicine(all)