. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 184 R. B. LOFTFIELD, E. A. EIGNER AND J. NOBEL LU M UJ 10 8 co 6 UJ TOADFISH VALINE ENZYME. ENZYME BLANK T T 10 20 30 MINUTES FIGURE 1. A typical experiment that shows the rate of valyl tRNA formation by the toad- fish valine activating enzyme with four different species of tRNA. The reactions were carried out at 25° with 10 mM ATP, M TRIS (pH ), 30 nM [14C]-valine, mg. tRNA per cc., and 6 jA. enzyme solution per tube (final vol. per tube 225 /*!.; four aliquots of 50 /id. eacli were taken at times indicated). The
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. 184 R. B. LOFTFIELD, E. A. EIGNER AND J. NOBEL LU M UJ 10 8 co 6 UJ TOADFISH VALINE ENZYME. ENZYME BLANK T T 10 20 30 MINUTES FIGURE 1. A typical experiment that shows the rate of valyl tRNA formation by the toad- fish valine activating enzyme with four different species of tRNA. The reactions were carried out at 25° with 10 mM ATP, M TRIS (pH ), 30 nM [14C]-valine, mg. tRNA per cc., and 6 jA. enzyme solution per tube (final vol. per tube 225 /*!.; four aliquots of 50 /id. eacli were taken at times indicated). The enzyme blank contains everything except added tRNA. The aliquots were worked up as previously described (Loftfield and Eigner, 1967a). The 30-minute observations correspond to the incorporation of m/j, mole of valine to 1 mg. of yeast tRNA and to m^ mole of valine into 1 mg. of starfish tRNA, exactly the same saturating values as found with homologous enzyme. On the other hand, the E. coli tRNA is only about one- tenth saturated. Hence the current work has been undertaken with considerable care to establish that aminoacylation of tRNA was actually taking place. Every figure noted in Table I represents a rate of reaction drawn from at least two different experiments in which the extent of reaction had been determined at no fewer than three time intervals. Figure 1 illustrates the kind of data used and the difficulties involved in comparing rates. For instance, many of our enzyme preparations contained an RNase which slowly destroyed the substrate RNA; this was clearly a greater problem if the reaction was slow or if larger amounts of enzyme solution were being used. Thus in some heterologous reactions similar to Figure 1, the tRNA was never saturated with amino acid. However, the addition of ATP and enzyme homologous with the tRNA at 30 minutes was ineffective. The receptor compe- tence of the tRNA had been destroyed during the incubation. This was especially evident
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