Evidence for leucylation of C.zeylanoides tRNA Ser CAG in vivo is notable that the K m values of the two pyrimidine At this point, we had established that the tRNA Ser CAG mutants, z-G33U (1.4 µM) and z-G33C (1.3 µM), are of C.zeylanoides is actually able to accept leucine in vitro. clearly lower than those of the two purine mutants, z-G33A However, considering the facts that SerRS and LeuRS (6.7 µM) and z-G33G (5.6 µM). The V max value of z-G33U coexist in cells and, judging from their K m values, that (1.2 pmol/min) is 39% of that of z-G33C (3.1 pmol/min), the affinity of tRNA Ser CAG toward SerRS is one order of which could explain why z-G33U shows lower leucylation magnitude higher than that toward LeuRS, we needed to activity than z-G33C despite having nearly the same K m ascertain whether the tRNA Ser CAG of C.zeylanoides is in value ( Figure 3B , right-hand graph). Judging from the fact leucylated in vivo. For this purpose, we adopted a sequence analysis (data not shown), the slight reduction newly developed method for quantifying an individual in the leucylation of z-G33G (5.6 µM) compared with aminoacyl-tRNA in cells (Suzuki et al., 1996) . that of the native tRNA Ser CAG (5.0 µM) is probably due Aminoacyl-tRNAs separately prepared from cells of to the partial deacetylation of ac 4 C at position 12, as C.zeylanoides and C.cylindracea were immediately submentioned above. This was confirmed by the observation jected to acetylation using [1-14 C]acetic anhydride to label of a slight reduction in leucylation activity also in acidthe amino acids as well as to stabilize the aminoacylated treated native tRNA Ser CAG (data not shown). It is thus tRNAs. From each of the acetylated aminoacyl-tRNA concluded that replacement of a pyrimidine by a purine mixtures, tRNA Ser CAGs from C.zeylanoides and C.cylindat position 33 has a repressive effect on leucylation of the racea were fished out by a solid-phase-attached DNA tRNA Ser CAG of C.zeylanoides.