Characterization of Aminoacyl Transfer Ribonucleic Acid Formation Stimulated by Polyamines

Abstract

From a comparative study of aminoacyl transfer ribonucleic acid (tRNA) formation stimulated by polyamines or by Mg2+, it is shown that both reactions have the same requirements for adenosine triphosphate, tRNA, and aminoacyl-tRNA synthetase. Reaction kinetics are similar in both reactions. The same tRNA is aminoacylated in the presence of either spermine of Mg2+. Aminoacyl-tRNA formed in the presence of polyamines can be a donor of amino acids in polypeptide synthesis. The Mg2+ requirement of aminoacyl-transfer ribonucleic acid (tRNA) formation can be replaced by polyamines, such as spermine, spermidine, and putrescine (8 to 20). In the aminoacyl-tRNA formation stimulated by polyamines, we have found that amino acid-dependent PPI-adenosine triphosphate (ATP) exchange, formation of amino acid hydroxamate, and formation of aminoacyl-adenosine monophosphate (AMP)-enzyme complex do not occur, although aminoacyl-tRNA formation does occur (7, 7a, 22). These findings suggest that polyamines cannot satisfy the Mg2+ requirement of the first reaction of aminoacylation: amino acid + ATP + enzyme aminoacyl-AMP-enzyme + PP,; but can satisfy the second reaction: aminoacyl-AMP-enzyme + tRNA = aminoacyl-tRNA + AMP + enzyme. If polyamines are ineffective in the first reaction, the question arises as to whether aminoacyl-tRNA formed in the presence of polyamines is the same as that formed in the presence of Mg2+. The present study was carried out to answer this question. MATERIALS AND METHODS Preparation and purification of aminoacyl-tRNA synthetase. Aminoacyl-tRNA synthetase (EC 6.1.1) was prepared as described previously (20). The enzyme was purified further by hydroxylapatite and diethylaminoethyl (DEAE)-Sephadex A-50 column chromatography. The hydroxylapatite IPresent address: Faculty of Pharmaceutical Sciences,

Trypsin modified isoleucyl transfer ribonucleic acid synthetase with partial retention of activity

“…These observations confirm the results reported by Piszkiewicz and Goitein (1974). Spermine stimulates aminoacylation of tRNA (Takeda et al, 1972). For isoleucyl-tRNA synthetase, the presence of 0.1 mM spermine tetrahydrochloride enhanced the charging activity of the native enzyme by 50%, but did not stimulate the charging activity of the modified enzyme.…”