MATERIALS AND METHODS

L-Tyrosine: tRNA ligase (AMP)[EC 6.1.1.1] has been purified to homogeneity from a genetically well-characterized, haploid strain of yeast, Saccharomyces cerevisiae aS288C. The kinetic constants for tRNATy are: tfra=3.5xl0-7M; £Cat=365mm-1. Under dissociating conditions, the enzyme gives a single band on discontinuous gel electrophoresis, corresponding to a molecular weight=31,500. Under non-dissociat-ing conditions, where the activity of the enzyme is retained, the molecular weight estimated by gel filtration is 116,000. Though tentative, these data indicate this enzyme may be composed of four subunits. Recently, we have found that crude enzyme preparations which are used widely to measure aminoacylation of tRNA contain a potent nuclease ( / , 2). This enzyme catalyzes the hydrolysis of the 3'-terminal AMP residue from normal tRNA. Even more extensive degrada-tion of the 3'-terminus occurs with tRNA that has been modified either with UV light or bisulfite ions, and it is not always possible to repair the degraded tRNA with the nucleo-tidyltransferase that is also present in the enzyme preparation. This nucleolytic activity seriously interferes with quantitative measure-ments of aminoacylation, and it can lead to misinterpretation of structure-action data. For this reason, we have undertaken the purifica-tion of several amino-acid: tRNA ligases from a genetically well-characterized, haploid strain of yeast, Saccharomyces cerevisiae aS288C (5). * Present address: Institute " Rudjer Boskovic," 41000 Zagreb, Yugoslavia. This paper describes the preparation of ho-mogeneous L-tyrosine: tRNA ligase (AMP) [EC 6.1.1.1] from this organism